Dendritic cell (DC)-based immunotherapy has been shown to be a promising strategy for anti-cancer therapy. Nevertheless, only a low overall clinical response rate has been observed in vaccinated patients with advanced cancer and therefore methods to improve DC immuno-stimulatory functions are currently under intense investigation. In this respect, we exposed human monocyte-derived DCs to a physiological temperature stress of 40°C for up to 24 h followed by analysis for (i) expression of different heat shock proteins, (ii) survival, (iii) cell surface maturation markers, (iv) cytokine secretion, and (v) migratory capacity. Furthermore, we examined the ability of heat-shocked DCs to prime naïve CD8(+) T cells after loading with MelanA peptide, by transfection with MelanA RNA, or by transduction with MelanA by an adenovirus vector. The results clearly indicate that in comparison to control DCs, which remained at 37°C, heat-treated cells revealed no differences concerning the survival rate or their migratory capacity. However, DCs exposed to thermal stress showed a time-dependent enhanced expression of the immune-chaperone heat shock protein 70A and both an up-regulation of co-stimulatory molecules such as CD80, CD83, and CD86 and of the inflammatory cytokine TNF-α. Moreover, these cells had a markedly improved capacity to prime autologous naïve CD8(+) T cells in vitro in an antigen-specific manner, independent of the method of antigen-loading. Thus, our strategy of heat treatment of DCs offers a promising means to improve DC functions during immune activation which, as a physical method, facilitates straight-forward applications in clinical DC vaccination protocols.